Mitochondria are central to the physiology of all eukaryotic cells. The immense diversity of mitochondria and their functions among the various branches of eukaryotic organisms is likely to have evolved in response to the diverse environmental niches of these organisms, which dictate the physiological demands placed on their mitochondria. The mitochondrion of malaria parasites has characteristics that are highly divergent from their hosts'. In the 1980s, our laboratory discovered the mitochondrial DNA (mtDNA) of malaria parasites. With its highly diminished gene content and organization, this genome presented the specter of divergent mitochondrial functions in Apicomplexan parasites that could be targets for novel antimalarial drugs. Previous studies from our laboratory have validated the parasite mitochondrion as a target for antimalarial drugs. The availability of genomic sequences and advances in gene transfer technology for malaria parasites has permitted us to explore various nuclearly encoded mitochondrial functions to assess their role in parasite physiology. Findings from this project have successfully addressed questions of long standing regarding the roles of major mitochondrial metabolic functions in P. falciparum: mitochondrial electron transport chain (mtETC), tricarboxylic acid (TCA) cycle, and heme biosynthesis. For the next funding period of this project, we wish to explore additional metabolic features of the parasite mitochondria that are essential for parasite survival and might be divergent from those in their mammalian counterparts. We have initiated experiments to derive a proteomic landscape of the parasite mitochondrion at different lifecycle stages through the use of a modified proximity biotinylation and allied approaches. In collaboration with the Sanger Institute we are identifying mitochondrially-targeted proteins, knockdown of which impacts blood stage growth of P. berghei. Genetic investigations combined with phenotypic studies involving these proteins will be carried out in P. falciparum. Several advances for genetic manipulation of P. falciparum, such as CRISPR-Cas9 editing and conditional knockdown of gene expression, have recently become available (and are currently used successfully in our laboratory), permitting relatively rapid gene knock-in/knockout as well as conditional knockdown mutant generation involving critical metabolic pathways. Genome scale disruption studies in P. beghei have also identified essential conserved un-annotated mitochondrially targeted proteins, functions of which will be investigated by conditional knockdown and phenotypic characterizations. Additional proteins to be investigated would be mitochondrial transport molecules, the mtETC components and the ATP synthase complex. Phenotypic characterization of these mutant parasites using various methods including metabolomics would bring our understanding of the unusual mitochondrial physiology of malaria parasite to an unprecedented level, which could inform future discoveries to control malaria.